CN117244114A - Biological tissue treatment method and artificial biological valve - Google Patents
Biological tissue treatment method and artificial biological valve Download PDFInfo
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- CN117244114A CN117244114A CN202210655398.7A CN202210655398A CN117244114A CN 117244114 A CN117244114 A CN 117244114A CN 202210655398 A CN202210655398 A CN 202210655398A CN 117244114 A CN117244114 A CN 117244114A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
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- Oral & Maxillofacial Surgery (AREA)
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention relates to a biological tissue treatment method and a prosthetic biological valve. The preparation method comprises the steps of firstly carrying out cell removal treatment on biological tissues, then carrying out crosslinking treatment on the biological tissues subjected to the cell removal treatment, and then carrying out aftertreatment on the crosslinked biological tissues by using aftertreatment liquid to obtain aftertreatment biological tissues, wherein the aftertreatment liquid comprises a biosurfactant and a prefabricated solvent. Compared with the prior art, the biological tissue treated by the post-treatment method has extremely low cytotoxicity and has remarkable anti-calcification effect in animals.
Description
Technical Field
The invention relates to the field of medical equipment, in particular to a biological tissue treatment method and a prosthetic biological valve.
Background
When a human valve is diseased, it can cause stenosis and regurgitation of the valve, causing heart valve disease, which is a major cause of morbidity and mortality worldwide. Over the past 10 years, transcatheter aortic valve implantation has matured gradually and has become the best solution for treating heart valve disease as clinical experience has accumulated through technological improvements and design iterations of transcatheter heart valves. To date, over 300,000 transcatheter aortic valve implants have been performed worldwide and it is expected that the number of 2050 will increase to 850000. Prosthetic heart valves are primarily mechanical and biological valves. The biological valve is mainly obtained by chemical treatment of biological valve of heterologous organism such as bovine pericardium, porcine pericardium, etc. Compared with mechanical valves, biological valves have the advantages of superior hemodynamic performance, no need of lifelong anticoagulation, low thromboembolism occurrence rate, strong anti-infective power and the like, and are widely paid attention to heart valve clinical operations.
However, biological valves have poor durability and a service life of only 10 years. The greatest cause of biological valve failure is calcification. Calcification of a biological valve means that calcium salt in the body is gradually deposited on the material of the biological valve after the biological valve is implanted into a human body to form calcified foci, so that the mechanical properties such as flexibility and the like of the biological valve are affected, and the failure of the valve is aggravated. The mechanism of calcification is quite complex and is not yet theorized. Most scholars believe that calcium phosphate accumulation is associated with toxicity of phospholipids, inflammatory cell infiltrates, mechanical damage, and residual materials in the cell membrane and in the cytoplasmic membrane. Currently, commercial valves in clinical use are essentially glutaraldehyde-cross-linked porcine or bovine pericardium. Glutaraldehyde has very excellent crosslinking performance, can obviously enhance the collagen stability and enzymolysis hydrolysis resistance of the components, and is still not replaced clinically.
However, although glutaraldehyde has outstanding properties in terms of crosslinking properties, it can significantly enhance the collagen stability and enzymatic hydrolysis resistance of the components, glutaraldehyde also has its naturally occurring drawbacks and shortcomings, and since glutaraldehyde has a certain biotoxicity, thus glutaraldehyde-treated prosthetic biological valves still have drawbacks of cytotoxicity, immune response, calcification, etc., which makes it often difficult to finally achieve the intended effect, despite the numerous technical means existing in the current anti-calcification field. This factor ultimately results in the service life of the prosthetic biological valve being a challenge.
Disclosure of Invention
Based on this, it is necessary to provide a method for treating biological tissue and a prosthetic biological valve to improve the biosafety and calcification resistance of the biological tissue and prosthetic biological valve.
The invention provides a method for treating biological tissues, which comprises the following steps:
performing decellularization treatment on biological tissues;
crosslinking the biological tissue subjected to the decellularization treatment;
and (3) performing post-treatment on the crosslinked biological tissue by using a post-treatment liquid to obtain a post-treated biological tissue, wherein the post-treatment liquid comprises a biosurfactant and a pre-prepared solvent.
In one embodiment, the biosurfactant comprises one or more of a saponin biosurfactant, a glycolipid biosurfactant, a lipopeptide biosurfactant, a polymer biosurfactant, a glycoside biosurfactant; the total mass concentration of the biosurfactant in the post-treatment liquid is 0.2-10% (wt/vol).
In one embodiment, the saponin biosurfactant comprises: one or more of ginsenoside, notoginsenoside, soyasaponin and digitonin; the glycolipid biosurfactant comprises: one or more of rhamnolipids, trehalose lipids, sophorolipids, mannosyl erythritol lipids; the lipopeptides biosurfactant comprises: one or more of surfactant, iturin, feng Yuan, subtilisin, lichenin; the polymeric biosurfactant includes: one or more of poly (N-p-vinylbenzyl maltoamide), polyhydroxystearate and its derivatives, alkyd polyethylene glycol and its derivatives, chitin/chitosan modified polymer; the total mass concentration of the biosurfactant in the post-treatment liquid is 0.2-1% (wt/vol).
In one embodiment, the temperature of the post-treatment is 25-37 ℃, the duration of the post-treatment is 5-12 hours, and the pH value of the post-treatment liquid is 6-9.
In one embodiment, the preformed solvent comprises an alcohol and a cross-linking agent, wherein the alcohol is one or more of isopropanol, ethanol, octanol, octanediol and 1-3 butanediol, and the volume concentration of the alcohol in the post-treatment liquid is 1-30% (vol/vol); the mass concentration of the cross-linking agent is 0.2-10% (wt/vol).
In one embodiment, the decellularizing treatment of the biological tissue comprises a step A, or the decellularizing treatment of the biological tissue comprises a step A and a step B; wherein,
step A: immersing the biological tissue in a solution containing a chemical detergent, and reacting for 6-24 hours under the conditions that the reaction temperature is 25-37 ℃ and the pH value is 6-9;
and (B) step (B): soaking the biological tissue obtained in the step A in a solution containing protease, and continuing to react for 6-24 hours under the conditions that the reaction temperature is 25-37 ℃ and the pH value is 6-9;
wherein the chemical detergent is one or more of sodium dodecyl sulfate, sodium deoxycholate, triton-100, tween-20, tween-80 and ethylenediamine tetraacetic acid, and the total weight concentration of the chemical detergent is 0.1-5% (wt/vol); the protease is one or more of DNase and RNase, and the concentration of the protease is 10-10000U/mL.
In one embodiment, the decellularizing agent is one or more of sodium deoxycholate and triton-100, and the biosurfactant is one or more of ginsenoside and surfactant; or the decellularized reagent is ethylenediamine tetraacetic acid with the mass concentration of 0.2-0.8%, and the biosurfactant is one or more of trehalose glycolipid and poly-N-p-vinylbenzyl maltoamide.
In one embodiment, the ratio of the mass concentration of the biosurfactant to the mass concentration of the chemical detergent is 1-2.
In one embodiment, the biological tissue is one or more of porcine pericardium, bovine pericardium, porcine aortic valve, swimming bladder.
The present invention also provides a prosthetic biological valve comprising post-treated biological tissue obtained according to the treatment method of any of the biological tissues described above.
According to the method for treating the biological tissue, the biological tissue subjected to the decellularization and crosslinking treatment is subjected to the aftertreatment by using the aftertreatment liquid, so that the residual chemical detergent and aldehydes in the decellularization and crosslinking treatment process can be sufficiently removed, the cytotoxicity of the aftertreatment biological tissue can be reduced, and the calcification resistance of the aftertreatment biological tissue can be improved. In addition, the post-treatment liquid can effectively dissolve the membrane-bound protein, simultaneously maintain the protein conformation in the solution phase, promote the re-overlapping after the protein denaturation, ensure that the shape of the collagen is kept complete, and is beneficial to improving the mechanical property of the post-treatment biological tissue. Therefore, the artificial biological valve prepared by using the post-treatment biological tissue has excellent biological safety, mechanical property and calcification resistance.
Drawings
FIG. 1 is a schematic flow chart of a method for treating biological tissue according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart of a method for treating biological tissue according to embodiment 1 of the present invention;
FIG. 3 is a graph showing the post-treatment group sample and the control group sample of example 1 of the present invention 30 days and 60 days after the wistar rats were implanted;
FIG. 4 is a histological stained image of alizarin red, masson and HE of the post-treatment group samples and the control group 2 samples of example 1 of the present invention 60 days after implantation of wistar rats.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
As shown in fig. 1, there is provided a method for treating biological tissue, comprising steps S10 to S30:
s10: the biological tissue is subjected to decellularization treatment.
In one embodiment, step S10 includes:
step S11, providing biological tissue to be treated.
The biological tissue comprises one or more of pig pericardium, cattle pericardium, horse pericardium, pig aortic valve, and swimming bladder. The biological tissue to be treated may be provided to be washed in advance, for example, the biological tissue may be washed clean with physiological saline at 4 ℃. The biological tissue is cleaned in advance, so that impurities can be removed.
Step S12, immersing the biological tissue to be treated in a decellularized reagent, and reacting for 6-24 hours under the conditions that the reaction temperature is 25-37 ℃ and the pH value is 6-9.
In one embodiment, the biological tissue may be subjected to a one-step decellularization process. In a specific example, the one-step decellularization process includes: immersing the biological tissue to be treated into a cell removing reagent containing chemical detergent with the total weight concentration of 0.1-5% (wt/vol), and reacting for 6-24 hours under the conditions that the reaction temperature is 25-37 ℃ and the pH value is 6-9; next, the biological tissue was rinsed with a buffer solution, and the rinsed biological tissue was stored in an antibacterial solution at a storage temperature of 4℃to obtain a decellularized biological tissue. The antibacterial solution may be PBS solution containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25 μg/mL) at a concentration of 1 g/L-10 g/L, or any other suitable solution with antibacterial effect. In this one-step decellularization process, the decellularization reagent used is a solution containing a chemical detergent. For example, the decellularizing agent is a buffer solution containing a chemical detergent, which is a solution obtained by dissolving one or more chemical detergents in a buffer solution.
The chemical detergent may be one or more of sodium dodecyl sulfate, sodium deoxycholate, triton-100, tween-20, tween-80 and ethylenediamine tetraacetic acid, for example, the chemical detergent may be ethylenediamine tetraacetic acid, a combination of sodium deoxycholate and triton-100, or a combination of other chemical detergents or a combination of multiple chemical detergents. The buffer used to flush the biological tissue may be any one or more of phosphate buffer (also known as PBS buffer), D-Hanks buffer, or piperazine ethane sulfonic acid solution (HEPES buffer). The specific preparation method of the PBS buffer solution comprises the following steps: 123g of disodium hydrogen phosphate heptahydrate, 22g of potassium dihydrogen phosphate, 73g of sodium chloride and 2g of potassium chloride are weighed, fully dissolved by 2L of sterile distilled water, and adjusted to the required pH by 1N hydrochloric acid and sodium hydroxide, and then fixed to 10L.
The total mass concentration of the chemical detergent is not too high, the biological tissue treated by the chemical detergent with high concentration is easy to be subjected to layering damage, and the residual chemical detergent on the surface of the biological tissue is possibly increased, so that the cytotoxicity and calcification amount of the biological tissue in the later stage are larger; however, too low a concentration of chemical detergent tends to result in insufficient decellularization treatment, and still more cell components remain; the total weight concentration of the chemical detergent is controlled to be in the range of 0.1% -5% (wt/vol).
In another embodiment, the biological tissue may be subjected to a step-wise decellularization process, i.e., multiple decellularization processes are performed on the biological tissue sequentially with multiple decellularization reagents. For example, after the above-described one-step decellularization treatment, the biological tissue is further treated with a decellularization reagent containing a protease. Wherein the protease is one or more of DNase and RNase, and the concentration of the protease is 10-10000U/mL. The decellularization reagent containing protease is a solution obtained by dissolving one or more proteases in Tris buffer, wherein the specific preparation method of the Tris buffer comprises the following steps: 1.576g Tris-HCl,2.380g magnesium chloride, 0.110g calcium chloride were weighed out in sterile distilled water and fixed to a volume of 1L. It should be noted that the buffer used in the present invention except protease is Tris buffer, and any of phosphate buffer (also referred to as PBS buffer), D-Hanks buffer or piperazine ethane sulfonic acid solution (HEPES buffer) may be used as the diluent or buffer of all other substances and solutions unless otherwise mentioned.
S20: and (3) performing cross-linking treatment on the biological tissue after the decellularization treatment.
In one embodiment, step S20 includes: immersing the biological tissue subjected to the decellularization treatment into a cross-linking agent solution for cross-linking. Wherein, the cross-linking agent can be glutaraldehyde or other aldehydes such as formaldehyde, succinaldehyde and the like, and the cross-linking agent solution is a solution obtained by dissolving aldehyde into a buffer solution.
In one embodiment, the mass concentration of aldehyde is 0.5-10%, the crosslinking temperature is 25-37 ℃, the crosslinking time is 2-30 days, and the pH value is 6-9.
It will be appreciated by those skilled in the art that any suitable cross-linking agent may be used in addition to the above exemplified and cross-linking agents, such as carbodiimide, quercetin, tannic acid, procyanidins, polyepoxides and the like.
S30: and (3) performing post-treatment on the crosslinked biological tissue by using a post-treatment liquid to obtain a post-treated biological tissue.
In one embodiment, step S30 includes: immersing the crosslinked biological tissue in a post-treatment liquid, wherein the post-treatment conditions comprise: the temperature is 25-37 ℃ and the duration is 5-12 h. Wherein the post-treatment liquid comprises a biosurfactant and a pre-prepared solution, and the pH value of the post-treatment liquid is 6-9.
In one embodiment, the biosurfactant comprises one or more of a saponin biosurfactant, a glycolipid biosurfactant, a lipopeptide biosurfactant, a polymer biosurfactant, and a glycoside biosurfactant.
Wherein the saponin biosurfactant comprises: one or more of ginsenoside, notoginsenoside, soyasaponin and digitonin.
Glycolipid biosurfactants include: one or more of rhamnolipids, trehalose lipids, sophorolipids, mannosyl erythritol lipids.
Lipopeptides biosurfactants include: one or more of surfactant, iturin, feng Yuan, subtilin, and lichenin.
The polymeric biosurfactant includes: one or more of poly (N-p-vinylbenzyl maltoamide), polyhydroxystearate and its derivatives, alkyd polyethylene glycol and its derivatives, and chitin/chitosan modified polymer.
The glycoside biosurfactant comprises: one or more of dodecyl beta-D-maltoside, dodecyl beta-D-glucopyranoside and octyl beta-D-glucopyranoside.
In one embodiment, the biosurfactant may be used singly or in combination of a plurality of biosurfactants.
Cytotoxicity is an important cause of affecting calcification, and different types of surfactants have different cytotoxicity, and generally, the order of toxicity is cationic > anionic > nonionic. The embodiment adopts a mild biosurfactant in the post-treatment process, has low cytotoxicity, can not bring additional influence to biological tissues, can remove residual chemical detergent on the surfaces of the biological tissues in the post-treatment process, reduces the cytotoxicity of the residual chemical detergent of the biological tissues in the decellularization process, and obviously improves the biocompatibility. In addition, the biosurfactant can effectively dissolve the membrane-bound protein, maintain the protein conformation in the solution phase and promote re-overlapping after protein denaturation. That is, the post-treatment process does not destroy the structure of the extracellular biological tissue of the biological valve, so that the biological valve maintains normal biological activity and function. The third post-treatment of the invention effectively removes lipid, stabilizes collagen and reduces cytotoxicity, ensures excellent crosslinking performance of glutaraldehyde, improves inherent cytotoxicity, immune response and calcification of glutaraldehyde, and the prepared artificial biological valve has extremely low cytotoxicity and biocompatibility and has remarkable anti-calcification effect in animals.
The concentration of the biosurfactant is not too high, the calcification resistance of the biosurfactant treated by the biosurfactant with high concentration is not enhanced, and the biosurfactant treated by the biosurfactant may be damaged; the biological surfactant with too low concentration is easy to cause insufficient combination with residual substances after cell removal treatment and crosslinking treatment, and the post-treatment effect is poor; so that the total mass concentration of the biosurfactant in the post-treatment liquid can be controlled to be 0.2-10%. For example, the concentration of the biosurfactant in the post-treatment liquid may be 0.2% to 1.0% by mass.
In one embodiment, the ratio of the concentration of the biosurfactant to the concentration of the chemical detergent is 1-2, so that the biosurfactant can be fully combined with the chemical detergent remained on the biological tissue, a better effect of removing residual substances on the biological tissue is realized, cytotoxicity is effectively reduced, and an anti-calcification effect is improved.
Further, in one of the embodiments, the chemical detergent is one or more of sodium deoxycholate and triton-100, and the biosurfactant is one or more of ginsenoside and surfactant. In another embodiment, the chemical detergent is ethylenediamine tetraacetic acid and the biosurfactant is one or more of trehalose lipids, poly-N-p-vinylbenzyl maltoamide. In the above embodiments, the biosurfactant used in the post-treatment step can more effectively remove the chemical detergent used in the decellularization process, thereby further reducing cytotoxicity of the surface of the biological tissue and improving calcification resistance of the biological tissue.
In one embodiment, the preformed solvent comprises an alcohol and a cross-linking agent, wherein the alcohol is one or more of isopropanol, ethanol, octanol, octanediol, 1-3 butanediol, and the volume concentration of the alcohol is 1-30% (vol/vol). The cross-linking agent comprises glutaraldehyde and other aldehydes, and the mass concentration of the cross-linking agent is 0.2-10%.
In one embodiment, in step S30, the method further includes a cleaning step after the crosslinked biological tissue is immersed in the post-treatment liquid: repeatedly cleaning the biological tissue by using a buffer solution to obtain the post-treatment biological tissue. For example, the biological tissue is repeatedly cleaned using PBS buffer.
The present invention also provides a prosthetic bioprosthetic valve prosthesis comprising an aortic valve, mitral valve, tricuspid valve, pulmonary valve, venous valve, etc.
In one embodiment, the prosthetic biological valve prosthesis comprises a valve stent and post-treated biological tissue produced by the treatment method of extracellular biological tissue described above.
In one embodiment, the post-treatment biological tissue is cut into a preset shape to form the valve leaflets, and the one or more valve leaflets are connected to the inside of the valve support by a suture or other connecting means, so that the post-treatment biological tissue has low toxicity and high calcification resistance, and is beneficial to improving the biological safety and the service life of the artificial biological valve.
In another embodiment, the post-treatment biological tissue is cut into a preset shape to form a skirt, and the formed skirt is connected and covered on the inner surface and/or the outer surface of the bracket in a connecting mode such as stitching, so that the perivalvular leakage can be effectively reduced, and the post-treatment biological tissue has low toxicity and high calcification resistance, so that the biological safety and the service life of the artificial biological valve are improved.
The following are specific examples.
In the following examples, the following test methods were used:
1. in vitro cytotoxicity experiments: in vitro cytotoxicity was tested according to ISO 10993-5:2009/GB/T16886.5, section 5 of medical device biological evaluation. The experiment adopts the CCK-8 method of leaching solution, cuts a sample into 1cm multiplied by 1cm, places the sample into a 24-hole plate, washes the sample with PBS after soaking and sterilizing the sample in 75% ethanol solution, and then adds a culture medium to culture the sample for 24 hours to obtain the productMaterial leaching liquor. HUVECs cells were cultured at 1X 10 4 The density of individual cells/wells was seeded in 96-well cell culture plates. After 24h of incubation, the original medium was discarded, the sample extract was changed, and incubation was continued for additional 24h and 72h. mu.L of 10% CCK-8 solution was added to the wells and incubated for 1 hour at 37 ℃. After discarding the leachate, 100. Mu.L of 10% CCK-8 solution was added to each well and incubated at 37℃for 1h. Finally, the absorbance at 450nm was measured for each sample with a microplate reader. The medium without sample served as a blank for cytotoxicity.
2. In vivo animal calcification and immune evaluation:
calcification assessment was performed using a rat subcutaneous implantation model. First, a sample was laser cut into 1cm×1cm and implanted under the skin of Wistar rats, taken out after 30 and 60 days of implantation, and a portion of the sample was used for drying, weighing and after they were digested in 6N HCl at 97 ℃ for 24 hours, the calcium content was measured using an inductively coupled plasma emission spectrometer (ICP-OES); a portion of the samples were fixed with 4% paraformaldehyde, paraffin embedded and sectioned, then stained with alizarin red, masson and HE, and immunohistochemical staining was performed with CD3, F4/80, TNF- α and IL-10 markers to analyze inflammation and immune response.
Example 1
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house was washed clean with physiological saline at 4deg.C, immersed in PBS solution containing 0.5% (wt/vol) Triton X-100 and 0.5% (wt/vol) sodium deoxycholate, and treated at 25deg.C for 12 hours; next, the sample was rinsed with PBS solution, soaked with 50U/mL deoxyribonuclease (DNase I) and 1000. Mu.g/mL ribonuclease (RNase), and the treatment was continued at 25℃for 12 hours. The protease dilutions were Tris buffer (10 mM Tris-HCl (pH=7.5), 2.5mM MgCl 2 ，0.1mM CaCl 2 ). The sample was then repeatedly rinsed with physiological saline. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.42) at room temperature of 25 ℃ to crosslink for 72 hours, to obtain crosslinked bovine pericardium. Will crosslink the bovine pericardium Immersed in a post-treatment solution, which was a PBS solution (ph=7.42) containing 0.5% (wt/vol) glutaraldehyde, 30% (vol/vol) isopropyl alcohol (IPA), and 0.5% (wt/vol) dodecyl- β -D-maltoside (DDM), and post-treated at 25 ℃ for 12 hours. Finally, the reagent residues were repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4), see in particular fig. 2.
Control group sample treatment: the treatment process of the sample in the control group 1 is different from that of the sample in the post-treatment group only in that the post-treatment liquid is different from that of the sample in the post-treatment group, and the post-treatment liquid used in the sample treatment of the control group does not contain dodecyl-beta-D-maltoside. The control group 2 samples differed from the post-treatment group samples only in the treatment procedure: the samples of control group 2 were not subjected to post-treatment.
Evaluation of results:
1. in vitro cytotoxicity experiments: the control group 2 samples had significantly less cell viability in HUVECS cell cultures for 1 day and 3 days than the post-treatment group. The comparison group 2 sample has obvious cytotoxicity, and cells basically die along with the extension of time, and the cell growth state of the post-treatment group is good and basically has no cytotoxicity.
2. In vivo animal calcification and immune evaluation: post-treatment and control sample patterns after 30 and 60 days of wistar rats implantation are shown in fig. 3, calcification evaluation is performed by an in vivo rat subcutaneous implantation model, and quantitative statistics of calcium ions are performed by ICP-OES. The control group 1 had a 30-day calcification of 45.24.+ -. 18.43. Mu.g/mg and a 60-day calcification of 93.95.+ -. 30.67. Mu.g/mg; the calcification amount of control group 2 was 90.59.+ -. 3.91. Mu.g/mg for 30 days and 138.61.+ -. 23.49. Mu.g/mg for 60 days; the calcification amount was only 0.77.+ -. 0.10. Mu.g/mg for 30 days and only 1.07.+ -. 0.15. Mu.g/mg for 60 days in the post-treatment group, with a significant difference. In addition, referring to fig. 4, tissue section staining was performed on a control sample implanted in rats for 60 days. The alizarin red staining clearly observed that the control group 2 sample was substantially completely calcified, the whole sample was stained with alizarin red vivid red, and the post-treatment group sample had no calcified area. The Masson dyeing can observe that the post-treatment group sample has obvious collagen staining, and the collagen fibers are in light blue complete and clear wavy shape, so that the collagen has complete structure, and stable elasticity and function. Whereas the control group 2 sample had no clear wavy streaks observed, and the collagen fibers had a large number of randomly oriented breaks. HE staining showed clear collagen fiber envelope and flap She Jiexian for post-treatment group samples, with substantially no inflammatory cell infiltration. And the fibrous capsule of the control group 2 sample is thickened, a large number of inflammatory cells are gathered outside the sample, and the immune rejection is serious. The results demonstrate the effectiveness of post-treatment groups to resist calcification and to preserve the structural and functional integrity of biological tissue.
Example 2
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 0.25% (wt/vol) sodium deoxycholate and 0.25% (wt/vol) triton-100, and treated at 37deg.C for 6 hr; next, the sample was rinsed with PBS solution, soaked with 10U/mL deoxyribonuclease (DNase I) and 2000. Mu.g/mL ribonuclease (RNase), and the treatment was continued at 37℃for 10 hours. The protease dilutions were Tris buffer (10 mM Tris-HCl (pH=7.5), 2.5mM MgCl 2 ，0.1mM CaCl 2 ). The sample was then repeatedly rinsed with physiological saline. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and crosslinked in 0.5% (wt/vol) glutaraldehyde PBS solution (ph=7.88) at 30 ℃ for 15 days to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 0.625% (wt/vol) glutaraldehyde, 20% (vol/vol) octanediol, 0.4% (wt/vol) ginsenoside and 0.4% (wt/vol) surfactant (pH=7.88), and post-treated at 37℃for 5 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
example 3
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 1.5% (wt/vol) sodium deoxycholate, and treated at 36deg.C for 10 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and crosslinked in 10% (wt/vol) glutaraldehyde PBS solution (ph=8.46) at 32 ℃ for 5 days to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 5% (wt/vol) glutaraldehyde, 5% (vol/vol) octanol, 2% (wt/vol) surfactant (pH=8.46), and post-treated at 28℃for 10 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
example 4
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, and immersed in PBS solution containing 0.1% (wt/vol) triton-100 for processing at 37deg.C for 6 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in a 3% (wt/vol) glutaraldehyde PBS solution (ph=7.7) at 35 ℃ to crosslink for 10 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 2% (wt/vol) glutaraldehyde, 20% (vol/vol) ethanol, 0.2% (wt/vol) ginsenoside (pH=7.0), and post-treated at 33℃for 8 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
example 5
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C, immersed in PBS solution containing 0.2% (wt/vol) ethylenediamine tetraacetic acid, and treated at 30deg.C for 24 hr; next, the sample was rinsed with PBS solution, soaked with 10000U/mL of deoxyribonuclease (DNase I) and 10. Mu.g/mL of ribonuclease (RNase), and the treatment was continued at 37℃for 6 hours. The protease dilutions were Tris buffer (10 mM Tris-HCl (pH=7.5), 2.5mM MgCl 2 ，0.1mM CaCl 2 ). The sample was then repeatedly rinsed with physiological saline. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in a 2% (wt/vol) glutaraldehyde PBS solution (ph=9.0) at 35 ℃ to crosslink for 30 days, to obtain crosslinked bovine pericardium. Immersing the cross-linked bovine pericardium in a post-treatment solution containing 1% (wt/vol) glutaraldehyde, 10% (vol/vol) isopropanol, 10% (vo)l/vol) ethanol, 0.2% (wt/vol) trehalose ester and 0.1% (wt/vol) poly-N-p-vinylbenzyl maltoamide in PBS (ph=9.0), and post-treated at 35 ℃ for 7 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
example 6
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 1.2% (wt/vol) ethylenediamine tetraacetic acid, and treated at 35deg.C for 12 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 5% (wt/vol) glutaraldehyde PBS solution (ph=6.0) at 37 ℃ to crosslink for 10 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 0.2% (wt/vol) glutaraldehyde, 10% (vol/vol) 1, 3-butanediol, 1.2% (wt/vol) trehalose lipid (pH=6.0), and post-treated at 30℃for 8 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
example 7
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 2.5% (wt/vol) ethylenediamine tetraacetic acid, and treated at 31deg.C for 10 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 8% (wt/vol) glutaraldehyde PBS solution (ph=7.3) at 37 ℃ to crosslink for 8 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 6% (wt/vol) glutaraldehyde, 25% (vol/vol) octanediol, 5% (wt/vol) poly-N-p-vinylbenzyl maltoamide (pH=8.2), and post-treated at 32℃for 6 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment. Evaluation of results:
In vivo animal calcification evaluation:
example 8
Post-treatment group sample treatment: fresh pig pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, and immersed in PBS solution containing 0.8% (wt/vol) Tween-20 for processing at 28deg.C for 10 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized porcine pericardium. The above decellularized pig pericarp was spread on a fixed plate to prevent curling, and placed in 10% (wt/vol) formaldehyde PBS solution (ph=8.1) at 28 ℃ to crosslink for 2 days, to obtain crosslinked pig pericarp. The crosslinked pig pericardium was immersed in a post-treatment solution of PBS containing 10% (wt/vol) formaldehyde, 1% (vol/vol) ethyloctanediol, 1.6% (wt/vol) rhamnolipid (pH=8.0), and post-treated at 29℃for 10 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated porcine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
In vivo animal calcification evaluation:
example 9
Post-treatment group sample treatment: fresh porcine aortic valves collected from slaughter houses were washed clean with physiological saline at 4℃and immersed in PBS solution containing 3% (wt/vol) sodium dodecyl sulfate for 20 hours at 32 ℃; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized porcine aortic valve. The decellularized porcine aortic valve was spread on a fixation plate to prevent curling, and placed in 9% (wt/vol) succinaldehyde PBS solution (ph=7.8) at 28 ℃ to crosslink for 20 days, to obtain a crosslinked porcine aortic valve. The crosslinked porcine aortic valve was immersed in a post-treatment solution of PBS solution (ph=8.5) containing 8% (wt/vol) succinaldehyde, 15% (vol/vol) 1,3 butanediol, 4.5% (wt/vol) digitonin, and post-treated at 27 ℃ for 11 hours. Finally, the reagent residue was repeatedly washed with PBS to give a post-treated porcine aortic valve and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
example 10
Post-treatment group sample treatment: washing fresh swimming bladder collected from slaughterhouse with 4 deg.C physiological saline, soaking in PBS solution containing 4% (wt/vol) Tween-80, and treating at 33deg.C for 15 hr; the samples were then repeatedly rinsed with PBS solution. The sample was then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) and stored at 4℃to give decellularized swimming bladder. The above cell-free swimming bladder was spread on a fixed plate to prevent curling, and placed in a 6% (wt/vol) glutaraldehyde PBS solution (ph=7.8) at 26 ℃ to crosslink for 5 days, to obtain a crosslinked swimming bladder. The crosslinked swimming bladder was immersed in a post-treatment solution of PBS containing 3% (wt/vol) formaldehyde, 18% (vol/vol) ethanol, 8% (wt/vol) lichenin (pH=7.9) and post-treated at 36℃for 9 hours. Finally, the reagent residue was repeatedly washed with PBS to give post-treated swim bladder, and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
Example 11
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 5% (wt/vol) sodium dodecyl sulfate, and treated at 26deg.C for 18 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 0.8% (wt/vol) glutaraldehyde PBS solution (ph=7.5) at 25 ℃ to crosslink for 25 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 0.3% (wt/vol) glutaraldehyde, 25% (vol/vol) isopropyl alcohol, 5% (wt/vol) polyhydroxystearate (pH=7.6), and post-treated at 29℃for 12 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
In vivo animal calcification evaluation:
comparative example 1
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 10% (wt/vol) sodium dodecyl sulfate, and treated at 26deg.C for 18 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 0.8% (wt/vol) glutaraldehyde PBS solution (ph=7.5) at 25 ℃ to crosslink for 25 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 0.3% (wt/vol) glutaraldehyde, 25% (vol/vol) isopropyl alcohol, 5% (wt/vol) polyhydroxystearate (pH=7.6), and post-treated at 29℃for 12 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4).
Evaluation of results: the post-treatment group samples of comparative example 1 exhibited delamination failure.
Comparative example 2
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 5% (wt/vol) sodium dodecyl sulfate, and treated at 26deg.C for 18 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 0.8% (wt/vol) glutaraldehyde PBS solution (ph=7.5) at 25 ℃ to crosslink for 25 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 0.3% (wt/vol) glutaraldehyde, 25% (vol/vol) isopropyl alcohol, 1% (wt/vol) polyhydroxystearate (pH=7.6), and post-treated at 29℃for 12 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4).
Evaluation of results:
in vivo animal calcification evaluation:
comparative example 3
Post-treatment group sample treatment: fresh bovine pericardium collected from slaughter house is washed with physiological saline at 4deg.C for 4 hr, immersed in PBS solution containing 5% (wt/vol) sodium dodecyl sulfate, and treated at 26deg.C for 18 hr; the samples were then repeatedly rinsed with PBS solution. The samples were then stored in PBS containing penicillin (100U/mL) -streptomycin (0.1 mg/mL) -amphotericin B (0.25. Mu.g/mL) at 4℃to give decellularized bovine pericardium. The above decellularized bovine pericardium was spread on a fixed plate to prevent curling, and placed in 0.8% (wt/vol) glutaraldehyde PBS solution (ph=7.5) at 25 ℃ to crosslink for 25 days, to obtain crosslinked bovine pericardium. The crosslinked bovine pericardium was immersed in a post-treatment solution of PBS containing 0.3% (wt/vol) glutaraldehyde, 25% (vol/vol) isopropyl alcohol, 10% (wt/vol) polyhydroxystearate (pH=7.6), and post-treated at 29℃for 12 hours. Finally, the reagent residue was repeatedly washed with PBS to obtain post-treated bovine pericardium and stored in 0.625% (wt/vol) glutaraldehyde PBS solution (ph=7.4). The control samples were without post-treatment. The control samples differ from the post-treatment samples only in the treatment procedure: samples of the control group were not subjected to post-treatment.
Evaluation of results:
in vivo animal calcification evaluation:
the post-treatment group samples of comparative example 3 had lower 30-day and 60-day calcification than the post-treatment group samples of comparative example 2.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A method of treating biological tissue, comprising: the method comprises the following steps:
performing decellularization treatment on biological tissues;
crosslinking the biological tissue subjected to the decellularization treatment;
And (3) performing post-treatment on the crosslinked biological tissue by using a post-treatment liquid to obtain a post-treated biological tissue, wherein the post-treatment liquid comprises a biosurfactant and a pre-prepared solvent.
2. The method of treating biological tissue according to claim 1, wherein the biosurfactant comprises one or more of a saponin biosurfactant, a glycolipid biosurfactant, a lipopeptide biosurfactant, a polymer biosurfactant, a glycoside biosurfactant; the total mass concentration of the biosurfactant in the post-treatment liquid is 0.2-10% (wt/vol).
3. The method for treating biological tissue according to claim 2, wherein the saponin biosurfactant comprises: one or more of ginsenoside, notoginsenoside, soyasaponin and digitonin; the glycolipid biosurfactant comprises: one or more of rhamnolipids, trehalose lipids, sophorolipids, mannosyl erythritol lipids; the lipopeptides biosurfactant comprises: one or more of surfactant, iturin, feng Yuan, subtilisin, lichenin; the polymeric biosurfactant includes: one or more of poly (N-p-vinylbenzyl maltoamide), polyhydroxystearate and its derivatives, alkyd polyethylene glycol and its derivatives, chitin/chitosan modified polymer; the total mass concentration of the biosurfactant in the post-treatment liquid is 0.2-1% (wt/vol).
4. The method according to claim 1, wherein the post-treatment is performed at a temperature of 25 to 37 ℃, the post-treatment is performed for a period of 5 to 12 hours, and the post-treatment solution has a pH of 6 to 9.
5. The method according to claim 1, wherein the pre-solvent comprises alcohols and a cross-linking agent, wherein the alcohols are one or more of isopropanol, ethanol, octanol, octanediol, and 1-3 butanediol, and the volume concentration of the alcohols in the post-treatment liquid is 1-30% (vol/vol); the mass concentration of the cross-linking agent is 0.2-10% (wt/vol).
6. The method of claim 2, wherein the decellularizing treatment of the biological tissue comprises a step a, or wherein the decellularizing treatment of the biological tissue comprises a step a and a step B; wherein,
step A: immersing the biological tissue in a solution containing a chemical detergent, and reacting for 6-24 hours under the conditions that the reaction temperature is 25-37 ℃ and the pH value is 6-9;
and (B) step (B): soaking the biological tissue obtained in the step A in a solution containing protease, and continuing to react for 6-24 hours under the conditions that the reaction temperature is 25-37 ℃ and the pH value is 6-9;
Wherein the chemical detergent is one or more of sodium dodecyl sulfate, sodium deoxycholate, triton-100, tween-20, tween-80 and ethylenediamine tetraacetic acid, and the total weight concentration of the chemical detergent is 0.1-5% (wt/vol); the protease is one or more of DNase and RNase, and the concentration of the protease is 10-10000U/mL.
7. The method according to claim 6, wherein the decellularizing agent is one or more of deoxycholate sodium and triton-100, and the biosurfactant is one or more of ginsenoside and surfactant; or the decellularized reagent is ethylenediamine tetraacetic acid with the mass concentration of 0.2-0.8%, and the biosurfactant is one or more of trehalose glycolipid and poly-N-p-vinylbenzyl maltoamide.
8. The method according to claim 6, wherein a ratio of a mass concentration of the biosurfactant to a mass concentration of the chemical detergent is 1 to 2.
9. The method of claim 1, wherein the biological tissue is one or more of porcine pericardium, bovine pericardium, porcine aortic valve, and swimming bladder.
10. A prosthetic biological valve, characterized in that it comprises a post-treated biological tissue obtained according to the method of treatment of a biological tissue according to claims 1-9.
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2022
- 2022-06-10 CN CN202210655398.7A patent/CN117244114A/en active Pending
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